1. Field of the Invention
The present invention relates to a glutamine synthetase knock out transgenic HEK293 (Human embryonic kidney 293) cell line and a production method of a target protein using the transgenic cell line.
2. Description of the Related Art
The size of the world-wide protein medicine market is as big as almost 15.7 billion dollars as of 2011. This market is expected to grow continuously to 20 billion dollars in 2016. Mammalian cells are the most widely used system in the production of biomedicine (antibody for treatment, growth factor, hormone, etc), which are exemplified by HEK293 cells, CHO (Chinese hamster ovary) cells, NS0 (mouse myeloma) cells, and PER.C6 (human retinal cells). Among them, CHO cells have been used for the production of ⅓ of the total animal cell originated protein medicine but there is some concern about the characteristics of the produced protein thereby because of the rodental characteristics of the cells.
The difference in the quality of the produced protein caused by such cell line origins is attributed to the posttranslational modification (PTM). In particular, the glycosylation process during which a sugar chain is added seems to have the biggest effect on the efficacy of the protein medicine product. The component and structure of a sugar chain attached onto a protein medicine have an effect on the persistence of an applied medicine and the medicinal effect of the same. When it is administered repeatedly, it can also be a reason of immunogenicity. The structure of the sugar chain adhering on the protein in CHO cells is the closest to that of human cells, compared with other rodent originated cells. However, it still contains such sugar chain structures that human cells do not contain which are N-glycosylneuraminic acid (Neu5Gc) and Galα1-3Galβ1-GlcNAc-R (α-Gal), and these sugar chains might cause an immune response, leaving a question in stability (Ghaderi et al., Biotechnol. Genet. Eng. Rev. 2012, 28:147-75). Therefore, it is necessary to develop a human originated production cell line that can be a useful system for the production of a protein that is equally qualified to the protein of human.
The human originated cell lines that have been used for the production of a protein medicine are HEK293, F2N, and PER.C6, etc. Among them, HEK293 cell line has a great potential for the production of a medicinal protein as a human originated host cell. And the reasons for that potential are as follows:                (1) HEK293 cell line has been most widely used in the field of science whose cellular characteristics have been continuously studied, so that the relevant information regarding the cell line is rich. (2) HEK293 cell line can be suspending-cultured in a serum free medium, so that it can be used for the high concentration cell culture and for the mass-production in an industrial scale. (3) The protein medicine produced in HEK293 cell line has been approved by such supervisory institutions as FDA and EMA, proving the stability of HEK293 as a production cell line. However in the real industrial field, HEK293 cell line is only applied for transient transfection system for the screening of a target protein for the protein production, and there is no stable protein production system for the continuous high concentration production established so far, suggesting that HEK293 is not widely used as expected for the mass production of a target protein as a real medicinal product.        
As a method for producing a protein medicine with high concentration using an animal cell line, a cell line selection technique using a gene amplification system has been developed. The most common systems are DHFR/MTX (dihydrofolate reductase/methotrexate) and GS/MSX (glutamine synthetase/methionine sulfoximine). These two systems are mainly used to produce a protein in CHO cell line. Particularly, a host cell is transfected with a plasmid comprising a therapeutic recombinant protein gene and a selection marker gene, and then a cell line is selected in an environment in the presence of an inhibitor corresponding to each enzyme gene. The transgenic cell line designed to be suitable for the production of a target protein amplified the selection marker gene included in the plasmid in order to overcome the added inhibitor, during which the therapeutic recombinant protein included together in the plasmid is also amplified. As a result, the cell line displaying a high productivity per cell (qp) can be selected, with which the continuous high concentration target protein production can be achieved. The DHFR/MTX and GS/MSX systems are widely used in the industry. In particular, GS/MSX system makes the selection of a high production clone possible simply by one time MSX amplification, so that the time for the cell line construction can be decreased with this system. Therefore, it is most widely used recently. A recombinant HEK293 cell line that can produce a target protein can be prepared by transfecting the HEK293 cell line with a therapeutic recombinant protein gene by using the GS/MSX system (Noh et al., Curr. Opin. Chem. Eng. 2013, 2:391-7).
In order for the gene amplification system to be effective, it is important to inhibit the effect of the selection marker gene included in the host cell in the course of the gene amplification mediated by an inhibitor. If a selection marker gene is already expressed at a high concentration in a host cell line, the endogenous selection marker gene is amplified together with the introduced protein production plasmid in the host cell despite an inhibitor is treated during the selection, so that the efficiency of the amplification of the therapeutic recombinant protein gene is accordingly reduced and the productivity per cell (qp) is arrested.
In the case of using the DHFR/MTX system, the selection marker gene dhfr knockout CHO dhfr(−) cell line has been developed. For the GS/MSX system, NSO or CHO-K1 cell line expressing the selection marker gene gs at a low level endogenously is used.
The present inventors noticed the high resistance against MSX, the GS inhibitor, in HEK293 cell line which was resulted from the endogenously, highly expressed GS protein (see FIG. 1). Therefore, the inventors confirmed that HEK293 cell line cannot be introduced in GS/MSX system without gene manipulation unlike the previous NS0 or CHO-K1 host cells. The present inventors introduced the gene editing method CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR/cas9, Cho et al., Nat. Biotechnol. 2013, 31(3): 230-2) targeting gs, the selection marker gene of gene amplification system in HEK293 cell line. As a result, the inventors constructed the glutamine synthetase (gs) gene knockout HEK293 cell line. Owing to the knockout of gs gene there, cell line selection can be efficiently performed by using MSX. The present inventors confirmed that the selection efficiency of a cell line for the production of a target protein was improved by increasing the concentration of MSX, leading to the completion of this invention.